The objective of our research proposal is to develop inhibitors of anthrax toxin that prevent the death of animals challenged with Bacillus anthracis spores. Anthrax toxin is a combination of three non-toxic proteins that are secreted separately by the bacterium and then assemble into toxic complexes on the mammalian cell surface. The protective antigen component of the toxin binds the anthrax toxin receptor and oligomerizes into heptamers that bind the toxic enzymes, edema factor and lethal factor. Inhibitors of anthrax toxin that block either toxin assembly or cytosolic delivery of the enzymatic proteins are expected to be effective anthrax therapeutics because the toxin is necessary for disease progression and causes death of the patient. We have previously synthesized a molecule consisting of multiple copies of a toxin-binding peptide coupled to a polymer backbone. The peptide alone can prevent the assembly of the tripartite toxin in vitro and its polyvalent display by the backbone increases its effective activity. We demonstrated that this inhibitor prevents the activity of anthrax toxin in rats, indicating that this molecule is a promising lead compound for an anthrax therapeutic. During the period of this proposal, we will test whether this inhibitor can prevent death of mice challenged with Bacillus anthracis spores. We will also assess the inhibitor's toxicity and pharmacokinetics. In addition to testing this lead compound, we will synthesize and test derivatives to develop a mature compound. The first class of molecules we synthesize will display inhibitory peptides from a variety a polymeric backbones and nanoparticles, which will be chosen for properties such as biocompatibility and bioavailability. The second class of molecules will consist of backbones of defined molecular weight that display multiple copies of the inhibitory peptide. We will also optimize the sequence of the peptide to increase its inhibitory activity. Reiterative design and pharmacological testing will facilitate the development of inhibitors with high potency, long lifetime, and low toxicity. These inhibitors may be an effective adjunct to antibiotic therapy in the treatment of anthrax.